DNA replication is an essential process that is regulated by conserved mechanisms throughout eukaryotes. Previous studies in the fission yeast S. pombe have been integral to the identification of the major components necessary for DNA replication. Two essential factors, Cdc18 and Cdt, have been shown to regulate DNA replication and are required for signaling the S phase checkpoint. I propose to evaluate the roles of Cdc18 and Cdt1 in both of these processes using genomic, genetic, and biochemical approaches. A genome-wide analysis of Cdc18 and Cdt1 chromatin binding will be performed, providing new insights into the requirements for these proteins in the regulation of the initiation of DNA replication as well as in their largely unstudied functions during S phase progression. Detailed studies of Cdc18 and Cdt1 binding to specific origins of replication will delineate the activities of both proteins required for different steps in replication and in the signaling of the S phase checkpoint. These studies will contribute to our understanding of DNA replication in fission yeast and throughout eukaryotes. Moreover, as cancer cells divide uncontrollably, it is critical to determine the fundamental mechanisms of cell cycle control and the alterations that may lead to cancerous cells. The experiments in this proposal are all important for understanding the onset of cancer and its treatment. [unreadable] [unreadable]